Mouse anti v5 antibody

For IPs, the S2 cells transfected with specific plasmids were lysed using 1 ml RIPA buffer (Merck, #20-188) containing complete protease inhibitor (Roche, #1697498001) and phosphatase inhibitor (Roche, #4906845001). The protein extracts were incubated with 30 μL HA antibody-coated magnet beads for 6 h at room temperature. The beads were collected and washed six times with lysis buffer, and then detected by western blot. For western blot, antibodies were used at the following concentrations for western blot: mouse anti-α-tubulin at 1:10,000 (Beyotime, #AF0001), mouse anti-V5 antibody at 1:4000 (Invitrogen, # R960-25), rabbit anti-pS6K at 1:1000 (Cell Signaling Technology, #9209), rabbit anti-HA antibody at 1:1000 (Beyotime, #AF0039), guinea pig anti-Nprl3 (generated at this work) at 1:2000.

Primary cortical neuronal cultures were prepared from P1 Sprague-Dawley rats as previously described (32 (link),34 (link)). For assessment of neurite length, cultures were co-transfected at DIV 3 with V5-tagged human VPS35 and GFP plasmids at a 10:1 DNA molar ratio (5 µg total DNA per 35 mm dish) using Lipofectamine 2000 reagent (Invitrogen). Cultures were fixed with 4% PFA at DIV 7 and immunostained with mouse anti-V5 antibody (Invitrogen), and anti-mouse IgG-AlexaFluor-633 antibody (Invitrogen). Images were acquired using an EVOS fluorescence digital microscope (Advanced Microscopy Group). ImageJ software was used to measure the length of the longest neurite from control (GFP-positive) and VPS35-expressing (GFP-/V5-positive) neurons. Sampling was performed randomly with ≥30 neurons measured per condition for each experiment from 4–6 independent cultures/experiments. All measurements were performed by investigators blinded to experimental condition. TUNEL labeling was performed as previously described (32 (link)) to assess apoptotic cell death in cortical neurons infected with lentiviral vectors expressing V5-tagged VPS35 or GFP at DIV 3 and fixed with 4% PFA at DIV 14. Cell viability was also assessed following treatment of lentiviral-infected cortical neurons seeded in 96-well plates with cellular toxins for 24 h using the alamarBlue Cell Viability assay (Invitrogen).

Transgenic fly lines were generated by BestGene Inc. using P-element mediated transformation. Crosses for rescue of the TCF/Pan embryonic phenotype were set up as indicated in Figure 6A. For the cuticle analysis, flies were allowed to lay eggs on grape juice plates for 6–8 hours at 25°C. Flies were then removed and plates were incubated for an additional 24–36 hours at 25°C. During this time a wet yeast paste was applied to the centre of each plate to attract hatching larvae and periodically removed leaving behind unhatched embryos. Unhatched embryos were collected and their cuticles were prepared as described previously [55] (link).
For the Western blots, flies were allowed to lay eggs for 4 hours and the embryos were incubated for an additional 6 hours, all at 25°C. Embryos were collected and dechorionated. They were then treated with hot SDS buffer and manually ground for 5 minutes. Samples were loaded into a gel. TCF-V5 was detected using mouse anti-V5 antibody (1∶5000, Invitrogen). Tubulin was used as a loading control. The ECL kit (Pierce) was used to visualize the blots.
TCF/Pan mutant alleles used were TCF² and TCF³. TCF² contains a base pair loss of ATT to AT leading to a frameshift at I106 and TCF³ contains a CAA to TAA mutation resulting in a stop codon at Q319 in the HMG domain [17] (link).

8-azido-ATP [γ] biotin and [α-³²P] 8-azido-ATP were synthetized by and purchased from Affinity Photoprobes (Lexington, KY); delta-aminolevulinic acid (dALA), reduced glutathione (GSH), oxidized glutathione (GSSG), ATP, ADP, AMP, CTP, GTP, TTP, anti-V5 agarose affinity gel beads and ExtrAvidin-peroxidase were from Sigma (Saint Louis, MO); Rabbit anti-ABCB10 mouse antibody (epitope FFDKTRTGELINRL) was made by Research Genetics, Inc. (Huntsville, AL); Mouse anti-V5 antibody was from Invitrogen (Carlsbad, CA); Rabbit anti-porin/VDAC mouse antibody was from Abcam (Cambridge, MA).

To visualize the subcellular localization of the transiently expressing POI, cells were plated on coverslips (thickness no. 1.5 and radius: 18 mm). For fixed cell imaging, cells were fixed with 4% paraformaldehyde and permeabilized with cold methanol for 5 min at −20 °C. Next, cells were washed with Dulbecco’s phosphate-buffered saline (DPBS) and blocked for 1 h with 2% BSA in DPBS at room temperature. To detect APEX2-fusion expression, cells were incubated with mouse anti-V5 antibody (Invitrogen, cat. no. R960-25, 1:5000 dilution) for 1 h at room temperature. After washing four times with TBST each 5 min, cells were simultaneously incubated with secondary Alexa Fluor 488-goat anti-mouse IgG (Invitrogen, cat. no. A-11001, 1:1000 dilution) and streptavidin–Alexa Fluor 568 IgG (Invitrogen, cat. no. S11226, 1:1000 dilution) for 30 min at room temperature. Cells were then washed four times with TBST each for 5 min. Immunofluorescence images were obtained and analyzed SP8 X, Leica (NICEM in Seoul National University, Korea) with objective lens (HC PL APO ×100/1.40 OIL), White Light Laser (WLL, 470–670 nm, 1 nm tunable laser), HyD detector, and controlled by LAS X software.